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Plaque assays have traditionally been a reliable way to determine the titer of a lytic virus. However, this method has several shortcomings in that it is time-consuming, labor intensive, and suffers from limited sensitivity. In this article, we describe a novel flow cytometry-based titration assay to quantify green fluorescent protein-labeled herpes simplex virus type 1 (HSV-1-GFP). Using this assay, we were able to directly quantify ten-fold dilutions of the virus in which every GFP-positive cell could be counted. In a head-to-head comparison with a traditional plaque assay, the flow cytometry assay showed a greater linear range and was accomplished in less than half the time of the plaque assay. Additionally, the cells prepared for flow cytometry could also be directly visualized by fluorescence microscopy. These results with HSV-1-GFP show proof of concept and are of practical use to herpesvirus researchers. Additionally, this technique could be easily modified to study other lytic or non-lytic viruses using antibodies against viral antigens...

Signaling Substances Used in Plant Defense: HPLC-MS/MS Analysis of Jasmonates

Plants must be capable of responding to climatic fluctuations, diurnal rhythms, available supplies of water and nutrients, and insect attacks and infestations. To ensure such responses, plants need a network of regulating substances called phytohormones. These substances enable plants to respond to both biotic and abiotic stresses by initiating a cascade of orchestrated actions, and to trigger development-specific processes. In this article, we will discuss a highly sensitive analytical method for quantitative determination of phytohormones. The main representatives of the plant hormones are jasmonic acid (JA), cytokines, auxins, abscisic acid, salicylic acid, gibberellins, and strigolactones...

Traditionally, the Six Sigma framework has underpinned quality improvement and assurance in biopharmaceutical manufacturing process management. This paper proposes a neural network (NN) approach to vaccine yield classification and compares it to an existing multiple linear regression approach. As part of the Six Sigma process, this paper shows how a data mining framework can be used to extract further value and insight from the data gathered during the manufacturing process, and how insights into yield classification can be used in the quality improvement process...

Influence of Cell Disruption Methods on the Recovery and Immunogenicity of a Fusion Protein for a Therapeutic Cancer Vaccine Against HPV

Two cell disruption methods, mechanical and chemical, were applied for the recovery of a fusion protein named CIGB 550-E7, expressed on Escherichia coli grown in defined saline media. A comparison of the methods was done, and various operating parameters for each technique were optimized to obtain the maximum disruption efficiency and CIGB 550-E7 protein release. The mechanical disruption’s yield and recovery were 1.24 and 1.37 times higher than those obtained with chemical disruption. Modified conditions were assayed for the CIGB 550-E7 obtained by chemically defined media using the mechanical and chemical cell disruption methods. The processes we developed allowed us to obtain an active pharmaceutical ingredient that fulfills the requirements stipulated by the regulatory authorities in terms of purity and lipopolysaccharide contaminants. In addition, the CIGB 550-E7 obtained from both methods showed similar biological activity so that either method could be used. Finally, a cost/benefit relationship (CBR) analysis was done for both disruption methods, and the CBR value for mechanical cell disruption demonstrated that this was the most feasible choice...

Biopharmaceutical Manufacturing: Current Titers and Yields in Commercial-Scale Microbial Bioprocessing

This article reports the average titers and yields currently attained with commercially manufactured biopharmaceuticals expressed by microbial systems such as E. coli and yeasts. A recent BioProcessing Journal article comparably covered results from the first phase of this study concerning historical titers and yields attained for commercial-scale biopharmaceutical production using mammalian cells (e.g., CHO). As with this prior mammalian component, public domain data concerning titers and yields attained with microbially manufactured products were obtained using all available sources. These included a review of available literature and direct contact with over 200 bioprocessing professionals identified as involved in relevant product research and development, and manufacturing. Unexpectedly, current microbial titers with commercially manufactured products were found to be consistent with those previously determined for mammalian products. However, purification yields attained with microbial manufacturing averaged only about 15%. This is much lower than with mammalian products yielding approximately 69%. Despite low downstream yields, microbial bioproduction continues to be considered less costly, simpler, faster, and generally more economical than mammalian manufacturing...

Direct, Real-Time Antibody-Based Quantification of Baculovirus

The impact of viruses—in geopolitical human health issues, in the production of vaccines and recombinant proteins, and in gene therapy and cancer treatments—highlights the need for a better understanding of the systems that are dependent upon them. A primary barrier to recognizing the full potential of these life-saving biomedical approaches is the scarcity of analytical methods capable of providing biologically relevant information without hindering the pace of development and production. ViroCyt® is a Colorado-based biotechnology company with one overriding focus: Enabling the rapid and specific quantification of viruses and virus-related particles. The ViroCyt Virus Counter® was designed to meet this objective. Until recently, the primary method employed by the Virus Counter for detecting virus was ComboDye, which utilized a general fluorogenic nucleic acid stain and a general fluorogenic protein stain to simultaneously label intact virus particles. The advantage of this approach is that it provides a broadly applicable reagent capable of quantifying many virus types. The disadvantage is that crude and/or complex matrices, in which there is high nucleic acid and/or protein background, can complicate the detection of individual virus particles. To address this issue, and to allow innovation to drive the technology into an expanded number of virus systems, ViroCyt recently created a second portfolio of reagents called ViroTag®. This technology employs fluorescently tagged antibodies specific for the virus of interest. The first members of this product line to be launched target two high-value viruses: baculovirus and human adenovirus. In this article, we focus on the application of the ViroTag system for rapid quantification during the bioprocessing of baculovirus-derived systems...